Chromosome studies in New Zealand species of Brachyscome (Asteraceae)

Abstract

Chromosome numbers were determined for plants of five species and seven additional entities of uncertain taxonomic status of Brachyscome. We confirmed the chromosome numbers of several species but found new numbers in plants related to B. humilis, B. montana and B. radicata. Karyotype analysis showed a common karyotype in the diploids and suggested that the polyploids are autopolyploids. There is some evidence of aneuploidy in plants related to B. sinclairii and B. radicata. Karyotype analysis suggests that structural chromosome changes may have accompanied polyploidy in tetraploid B. humilis.

Keywords:

• Asteraceae
• Brachyscome
• chromosome number
• polyploidy
• New Zealand flora

The genus Brachyscome Cass. (=Brachycome orth. var. Cass.) in the Asteraceae is amongst the chromosomally most variable genera of angiosperms (Weiss-Schneeweiss & Schneeweiss 2013). In Australia, where the majority of species occur, there are species with 15 different chromosome numbers that range from 2n=4 to 2n=36, large differences in total chromosome length and also in the degree of karyotype asymmetry (Smith-White et al. 1970; Watanabe et al. 1999). In New Zealand, Webb et al. (1988) recognize five endemic species and more recently, de Lange & Rolfe (2010) recognize seven species, two of which, B. humilis G.Simpson & J.S.Thomson and B. radicata Hook.f., they conclude are species aggregates. A recent phylogenetic analysis placed the New Zealand endemic B. humilis within the same clade as the Australian species which supports the idea that the New Zealand species have arrived by long distance dispersal from Australia (Brouillet et al. 2009).

Previous observations on the chromosomes of the New Zealand species reported a range of numbers from 2n=18 to 2n=90 (summarized in Table 1). Brachyscome humilis is reported to have two chromosome numbers, 2n=36 and 2n=37 (Beuzenberg & Hair 1984; Dawson & Beuzenberg 2000); an interesting observation as examples of intraspecific chromosome number variation are relatively rare (< 3% of species) in the New Zealand flora (de Lange et al. 2008; Murray & de Lange 2013).

Table 1  Previously published chromosome numbers in the New Zealand species of Brachyscome.

Species	2n	Reference
B. humilis G.Simpson & J.S.Thomson	36, 37	Beuzenberg & Hair (1984); Dawson & Beuzenberg (2000)
B. linearis (Petrie) Druce	18	Dawson & Beuzenberg (2000)
B. longiscapa G.Simpson & J.S.Thomson	18	de Lange et al. (2004)
B. pinnata Hook.f.	18	Beuzenberg & Hair (1984) as B. sinclairii var. pinnata; de Lange & Murray (2002)
B. radicata Hook.f.	c.90	Dawson & Beuzenberg (2000)
	90	Hair in Smith-White et al. (1970);
		Beuzenberg & Hair (1984); de Lange & Murray (2002)
B. sinclairii Hook.f.	18	Beuzenberg & Hair (1984) as B. sinclairii var. sinclairii; Dawson & Beuzenberg (2000)

Because B. humilis and B. radicata are considered to be species aggregates (de Lange & Rolfe 2010), in this paper we have analysed the chromosomes of plants matching the types of both species as well as morphologically divergent plants believed to be allied to them. In addition, we include several plants believed to be allied to B. sinclairii Hook.f. and B. montana G.Simpson. The taxonomic status of New Zealand species of Brachyscome is currently under review and this paper is a contribution toward the review describing the results of our cytological investigation of the genus.

Materials and methods

The plant material used in this study is listed in Table 2. All plants were collected as living specimens from the wild. Somatic chromosomes were obtained from root tip meristems that were pretreated with a saturated solution of paradichlorobenzene for c. 18 h at 4 °C, fixed in ethanol/acetic acid (3:1 v/v), hydrolysed in 1 M HCl for 8 min before staining with FLP orcein. A minimum of five cells per plant were observed and counted.

Table 2  Chromosome counts, collection details, Landcare Research Garden number and herbarium voucher number for the Brachyscome plants studied.

Species	2n	Source	Garden number	Voucher number
Brachyscome longiscapa	18	South I., Otago, Awahokomo,Waitaki Valley	–	AK 233220
	18	South I., Otago, Ohau Downs, L. Ohau	G208/97	CHR 621909
B. pinnata	18	South I., Canterbury, Medbury Reserve	G132/95	CHR 621910
	18	South I., Canterbury, Medbury Reserve	G124/95	CHR 621911
	18	South I., Canterbury, L. Roundabout	G295/99	CHR 621912
	18	South I., Canterbury, Lees Valley	G118/01	CHR 621913
B. aff. sinclairii	18	South I., Marlborough, Flaxbourne River	G14292	CHR 621914
B. sp. (indet.)	18	South I., Otago, Rock & Pillar Ra.	G212/97	CHR 621915
	18	South I., Otago, Little Valley, Alexandra	G32/96	CHR 621916
B. aff. humilis	18	South I., Southland,West Dome	G263/98	CHR 621917
B. aff. montana	18	South I., Otago, The Remarkables, Doolans Creek	G200/97	CHR 621918
B. sp. (indet.)	18	South I., Marlborough, Ward Beach limestone	G131/92	CHR 621919
B. aff. sinclairii	28	South I., Marlborough, Ward Beach limestone	G148/00	CHR 621920
B. montana	35/36	South I., Otago, Mt.Cardrona	G210/97	CHR 621921
B. humilis	36	South I., Otago, Rock & Pillar Ra.	G18306	CHR 621922
B. sp. (indet.)	36	South I., Southland, Livingstone Mts	G44/00	CHR 621923
B. radicata	90	North I., South Auckland, Kaweka Ra.	G66/9	CHR 621924
B. aff. radicata	92	South I., Southland, Umbrella Mts	G497/98	CHR 621925

Results

We found five different chromosome numbers in the 18 plants that we analysed. Twelve plants were diploid with 2n=18, one had 2n=28, three were tetraploids with 2n=36 (though one plant, the sole example of B. montana s.s. [G210/97, Fig. 1G] appeared to contain cells with 35 or 36 chromosomes), one was decaploid with 2n=90 (Fig. 1J) and one was decaploid but had two extra chromosomes (2n=92, Fig. 1K) (Table 2 and Fig. 1). Karyotypes were constructed for three plants, the diploid B. longiscapa G.Simpson & J.S.Thomson (Fig. 2A), B. aff. sinclairii (G148/00, Fig. 2B) with 2n=28 and tetraploid B. humilis (G18306) (Fig. 2C). The diploid karyotype comprised eight small metacentric or sub-metacentric chromosomes and ten large, acrocentric chromosomes (Fig. 2A). All the diploid plants that we examined had a similar assortment of chromosomes. The majority of chromosomes of the B. aff. sinclairii plant (G148/00) can be arranged into sets of three presumed homologues with one additional small chromosome (Fig. 2B). In the tetraploid B. humilis most, although possibly not all, of the chromosomes can be arranged in sets of four, but as shown in Fig. 2C there appears to have been some chromosome rearrangement because not all presumed homologues can be arranged in groups of four. However, we appreciate that the placing of the chromosomes into groups of presumed homologues is somewhat subjective but it does appear in this plant that not all the chromosomes can be placed into groups of four. In B. radicata we have confirmed previous counts of 2n=90 in one plant and found 2n=92 in another that is allied to B. radicata. This latter plant is either an aneuploid or has two B chromosomes, we cannot confirm at this stage which of these possibilities is correct.

Figure 1  Mitotic metaphase in New Zealand Brachyscome. A, B. longiscapa 2n=18 (AK 233220). B, B. pinnata 2n=18 (G132/95). C, B.aff. sinclairii 2n=18 (G14292). D, B.sp. (indet.) 2n=18 (G212/97). E, B.aff. montana 2n=18 (G200/97). F, B. aff. sinclairii 2n=28 (G148/00). G, B. montana 2n=36 (G210/97). H, B. humilis 2n=36 (G18306). I, B. sp. (indet.) 2n=36. J, B. radicata 2n=90 (G66/9). K, B.aff. radicata 2n=92 (G497/98). Scale bar = 10 µm.

Figure 2  The karyotypes of three Brachyscome species of different ploidy levels. A, B. longiscapa 2n=18 (AK 233220). B, B. aff. sinclairii 2n=28 (G148/00). C, B. humilis 2n=36 (G18306). The chromosomes of B. humilis that do not appear to have obvious homologues are indicated by ★.

Discussion

We have confirmed previous chromosome counts for many of the New Zealand species of Brachyscome and found diploid numbers in two of the undetermined Brachyscome from Otago (G212/97 & G32/96) and one from Marlborough (G131/92). We found one tentatively new species currently allied to B. humilis (G263/98) had 2n=18, rather than the expected 2n=36 (Beuzenberg & Hair 1984; Dawson & Beuzenberg 2000). One plant morphologically allied to B. sinclairii had 2n=28 but the other plant had 2n=18, the same number as previously reported for B. sinclairii. In B. radicata, one plant had the expected number of 2n=90 but the other, which is similar to that species, had 2n=92. Neither 2n=28 nor 2n=92 have been reported previously in New Zealand species of the genus (Table 1). Our 2n=35/36 count for B. montana G.Simpson is the first for this species and the B.aff. montana plant from The Remarkables was diploid. Described by Simpson (1945) B. montana was treated as ‘Incertae Sedis’ by Allan (1961, p. 604–605) and the species was not discussed by Webb et al. (1988) although it was accepted as distinct by Druce (1993), Webb & Simpson (2001) and de Lange & Rolfe (2010). Of the two plants we counted, one (G210/97) comes from the type locality (Mt Cardrona, Central Otago) and matches Simpson's 1945 protologue as to description. The second plant (G200/97), from Doolan's Creek, The Remarkables, Central Otago is also a very close match to Simpson's description of B. montana but is clearly diploid. At this stage it is not clear whether there are diploid and tetraploid races of B. montana or whether the diploid plant represents another, potentially distinct segregate from B. montana. Central to this problem is that the type of B. montana appears to be missing (Allan 1961), and until the typification of B. montana is resolved any further taxonomic conclusions with respect to our diploid count would be premature.

Unlike in so many New Zealand native plants where chromosomes are small and often numerous, the chromosomes of Brachyscome are relatively large, in most cases relatively few in number and in addition individual chromosome morphology and size is variable. This makes basic karyotype analysis possible and this offers the potential to elucidate whether karyotype changes have accompanied speciation. All of our diploids appear to have the same basic karyotype and this is similar to a karyotype, presented as an idiogram, for a further undescribed entity (CHR 518417) from Ward Beach, Marlborough (Dawson & Beuzenberg 2000). Our undescribed plant from the Livingstone Mountains in Southland had a tetraploid number, the same number that we found in a plant, collected as B. humilis, from the Rock and Pillar Range, Otago. The karyotype of this Otago plant showed many chromosomes in groups of four, suggestive of autopolyploidy, though there are possibly some chromosomes in the complement that are not easily grouped in that way, thus suggesting that some structural rearrangements may have occurred associated with whole genome duplication. The plant with 2n=28 from Ward Beach appears to be an autotriploid, as those of its chromosomes that can be readily distinguished fall into groups of three apparent homologues, but it must also be aneuploid for one of the small chromosomes which we cannot differentiate unambiguously one from another. The plant of B.aff. radicata from the Umbrella Mountains also appears to be an aneuploid, although in this case of a decaploid, or to contain two B chromosomes. Aneuploids are not commonly found in natural plant populations but they are not unknown and a study of the rare Australian daisy, Rutidosis leptorrhynchoides F.Muell. as well as the New Zealand native Crassula ruamahanga A.P.Druce found extensive aneuploidy variation in several wild populations (Murray & Young 2001; de Lange et al. 2008).

The New Zealand species of Brachyscome, like their Australian congeners, are chromosomally variable showing evidence of both euploid and aneuploid variation. At this stage it is unclear whether any of this chromosome variation underlies the apparent taxonomic variation. The genus in New Zealand needs further detailed study at the population level to clarify the extent of chromosome variation and the application of molecular cytogenetic analyses to better understand genome evolution in the group. Further investigation is also needed to clarify the systematic position of the variants of the named species that we have studied.

Acknowledgements

We thank Nick Head, Cathy Jones and Neill Simpson for assistance in the field and David Purcell (Landcare Research, Lincoln Campus) for tending the plants examined in this study.